Nanoscale organization and dynamics of the siglec CD22 cooperate with the cytoskeleton in restraining BCR signalling.

Receptor organization and dynamics at the cell membrane are important factors of signal transduction regulation. Using super-resolution microscopy and single-particle tracking, we show how the negative coreceptor CD22 works with the cortical cytoskeleton in restraining BCR signalling. In naïve B cells, we found endogenous CD22 to be highly mobile and organized into nanodomains. The landscape of CD22 and its lateral diffusion were perturbed either in the absence of CD45 or when the CD22 lectin domain was mutated. To understand how a relatively low number of CD22 molecules can keep BCR signalling in check, we generated Brownian dynamic simulations and supported them with ex vivo experiments. This combined approach suggests that the inhibitory function of CD22 is influenced by its nanoscale organization and is ensured by its fast diffusion enabling a "global BCR surveillance" at the plasma membrane.

Cdc42 is a key regulator of B cell differentiation and is required for antiviral humoral immunity.

The small Rho GTPase Cdc42, known to interact with Wiskott-Aldrich syndrome (WAS) protein, is an important regulator of actin remodeling. Here, we show that genetic ablation of Cdc42 exclusively in the B cell lineage is sufficient to render mice unable to mount antibody responses. Indeed Cdc42-deficient mice are incapable of forming germinal centers or generating plasma B cells upon either viral infection or immunization. Such severe immune deficiency is caused by multiple and profound B cell abnormalities, including early blocks during B cell development; impaired antigen-driven BCR signaling and actin remodeling; defective antigen presentation and in vivo interaction with T cells; and a severe B cell-intrinsic block in plasma cell differentiation. Thus, our study presents a new perspective on Cdc42 as key regulator of B cell physiology.

The actin and tetraspanin networks organize receptor nanoclusters to regulate B cell receptor-mediated signaling.

A key role is emerging for the cytoskeleton in coordinating receptor signaling, although the underlying molecular requirements remain unclear. Here we show that cytoskeleton disruption triggered signaling requiring not only the B cell receptor (BCR), but also the coreceptor CD19 and tetraspanin CD81, thus providing a mechanism for signal amplification upon surface-bound antigen stimulation. By using superresolution microscopy, we demonstrated that endogenous IgM, IgD, and CD19 exhibited distinct nanoscale organization within the plasma membrane of primary B cells. Upon stimulation, we detect a local convergence of receptors, although their global organization was not dramatically altered. Thus, we postulate that cytoskeleton reorganization releases BCR nanoclusters, which can interact with CD19 held in place by the tetraspanin network. These results not only suggest that receptor compartmentalization regulates antigen-induced activation but also imply a potential role for CD19 in mediating ligand-independent "tonic" BCR signaling necessary for B cell survival.

F-actin is an evolutionarily conserved damage-associated molecular pattern recognized by DNGR-1, a receptor for dead cells.

Sterile inflammation can be initiated by innate immune recognition of markers of tissue injury termed damage-associated molecular patterns (DAMPs). DAMP recognition by dendritic cells (DCs) has also been postulated to lead to T cell responses to foreign antigens in tumors or allografts. Many DAMPs represent intracellular contents that are released upon cell damage, notably after necrosis. In this regard, we have previously described DNGR-1 (CLEC9A) as a DC-restricted receptor specific for an unidentified DAMP that is exposed by necrotic cells and is necessary for efficient priming of cytotoxic T cells against dead cell-associated antigens. Here, we have shown that the DNGR-1 ligand is preserved from yeast to man and corresponds to the F-actin component of the cellular cytoskeleton. The identification of F-actin as a DNGR-1 ligand suggests that cytoskeletal exposure is a universal sign of cell damage that can be targeted by the innate immune system to initiate immunity.

B cell receptor-mediated antigen gathering requires ubiquitin ligase Cbl and adaptors Grb2 and Dok-3 to recruit dynein to the signaling microcluster.

The B cell receptor (BCR) mediates B cell antigen gathering and acquisition for presentation to T cells. Although the amount of antigen presentation to T cells determines the extent of B cell activation, the molecular mechanisms underlying antigen gathering remain unexplored. Here, through a combination of high-resolution imaging, genetics and quantitative mass spectrometry, we demonstrate that adaptors Grb2 and Dok-3, and ubiquitin ligase Cbl in signaling BCR microclusters mediate association with the microtubule motor dynein. Furthermore, we visualize the localization and movement of these microclusters on the underlying microtubule network. Importantly, disruption of this network or diminished dynein recruitment in Grb2-, Dok-3-, or Cbl-deficient B cells, does not influence microcluster formation or actin-dependent spreading, but abrogates directed movement of microclusters and antigen accumulation. Thus we identify a surprising but pivotal role for dynein and the microtubule network alongside Grb2, Dok-3, and Cbl in antigen gathering during B cell activation.